Phosphorylation of phase‐separated p62 bodies by ULK1 activates a redox‐independent stress response

Abstract NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox‐dependent manner. p62 bodies formed by liquid–liquid phase separation contain Ser349‐phosphorylated p62, which participates in the redox‐independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1‐dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62 S351E/+ mice are phosphomimetic knock‐in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62 S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1‐knockout mice. Our results expand our understanding of the physiological importance of the redox‐independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.


Introduction
Liquid-liquid phase-separated biomolecular condensates, liquid droplets play an important role in many biological processes, such as gene expression, protein translation, stress response, and protein degradation, by incorporating a variety of RNA and client proteins into their interior depending on the intracellular context (Alberti & Hyman, 2021). Autophagy is involved in the degradation of several cytoplasmic liquid droplets, including stress granules and P bodies, and defects in this process are thought to cause transition of these droplets to the solid phase, resulting in the development of intractable diseases such as neurodegenerative disorders and cancer (Woodruff et al, 2018;Noda et al, 2020). Of the droplets that have a unique biological function and are degraded by autophagy, p62 bodies (also called p62 droplets) are liquid droplets formed by liquidliquid phase separation (LLPS) of p62 and its binding partners, ubiquitinated proteins (Sun et al, 2018;Zaffagnini et al, 2018). p62 bodies are involved in the regulation of intracellular proteostasis through their own autophagic degradation, and also contribute to the regulation of the major stress-response mechanism by sequestration of a client protein, kelch-like ECH-associated protein 1 (KEAP1) (Kirkin & Rogov, 2019;Faruk et al, 2021b).
Unc-51-like kinase 1 (ULK1) phosphorylates p62 at Ser407, inhibiting dimer formation of the ubiquitin-associated (UBA) domain of p62 (Isogai et al, 2011;Lim et al, 2015), and subsequent phosphorylation of Ser403 by TBK1, CK2, TAK1, and ULK1 allows binding of ubiquitinated proteins (Matsumoto et al, 2011;Pilli et al, 2012;Lim et al, 2015;Kehl et al, 2019). These phosphorylation events are thought to promote LLPS (Sun et al, 2018;Zaffagnini et al, 2018). In the degradation of p62 bodies, the ULK1 protein kinase complex consisting of FIP200/RB1-inducible coiled-coil protein 1 (hereafter FIP200), ULK1, ATG13, and ATG101 is translocated onto the bodies by binding of the FIP200 Claw domain to p62 (Turco et al, 2019). Alternatively, the ULK1 protein kinase complex is recruited to p62 bodies through the interaction of FIP200 with TAX1BP1, which localizes at p62 bodies through the interaction with the p62 binding partner NBR1 (Turco et al, 2021). Subsequently, ATG proteins assemble around the bodies (Kageyama et al, 2021). In the end, the p62 bodies are surrounded by autophagosomes due to the wetting effect (Agudo-Canalejo et al, 2021) and the binding of LC3 or GABARAP to p62 on the isolation membrane (Kageyama et al, 2021), followed by lysosomal degradation.
KEAP1 is an adaptor protein of cullin 3 ubiquitin ligase for nuclear factor (erythroid-derived 2)-like 2 (NRF2), which is a key transcription factor for a series of genes encoding anti-oxidative proteins and enzymes (Yamamoto et al, 2018). In the canonical pathway, KEAP1 is inactivated by the modification of oxidants, and NRF2 is then activated via redox-dependent regulation (Yamamoto et al, 2018). This redox-dependent pathway has been shown to be important in redox, metabolism, and protein homeostasis, as well as in the regulation of inflammation and cellular protection against many pathological conditions (Yamamoto et al, 2018;Cuadrado et al, 2019). In addition to this canonical pathway, a specific region of p62 directly interacts with KEAP1, competitively preventing the interaction between KEAP1 and NRF2 (Komatsu et al, 2010). The phosphorylation of Ser349 located in the KEAP1-interacting region of p62 enhances the interaction of p62 with KEAP1, resulting in full activation of NRF2 independently of redox conditions (Ichimura et al, 2013). However, the kinase(s) and regulatory mechanism underlying the redox-independent pathway, as well as its physiological significance, remain unclear.
Herein, we show for the first time that ULK1 is a major kinase for Ser349 of p62, both in vitro and in vivo. ULK1 directly interacts with p62 and phosphorylates Ser349 of p62. ULK1 localizes in p62 bodies in vitro and in vivo in a FIP200-independent fashion. While this phosphorylation does not affect the influx of KEAP1 into p62 bodies, it inhibits KEAP1 outflow, keeping KEAP1 in the p62 bodies and activating NRF2. Knock-in mice with a phosphomimetic mutation, but not those with a phosphodefective mutation, exhibit persistent activation of NRF2, which causes hyperkeratosis and consequently obstruction of the esophagus and forestomach, and eventually severe growth retardation due to malnutrition. Taken together, these results indicate the physiological importance of the p62 body-and ULK1-dependent and redox-independent stress response.

ULK1 directly interacts with and phosphorylates p62
To clarify whether the ULK1 kinase itself has an effect on the physical properties and physiological role of p62 bodies, we first studied the physical interaction of p62 with ULK1 or its yeast homolog Atg1 using high-speed atomic force microscopy (HS-AFM) (Fig 1). ULK1 has a serine-threonine kinase domain (KD) at the N-terminus and two microtubule interaction and transport (MIT1 and 2) domains at the C-terminus, all of which are conserved between yeast and mammals ( Fig 1A). The KD and MIT1/2 domains are linked by an intrinsically disordered region (IDR) (Fig 1B). p62 contains an N-terminal Phox1 and Bem1p (PB1) domain and a C-terminal UBA domain, as well as several interacting regions such as the LC3-interacting region (LIR) and KEAP1-interacting region (KIR), located in an IDR between the PB1 and UBA domains (Fig 1A and B). We purified recombinant p62 (268-440 aa and 320-440 aa) and SNAP-tagged ULK1 and Atg1 (Fig 1C). HS-AFM of SNAP-ULK1 revealed that like Atg1, it contains two globular domains consisting of a KD and two tandem MIT domains, linked to each other with an IDR, (Kodera et al, 2021) (Fig EV1A, Movie EV1). Meanwhile, HS-AFM of p62 (268-440 aa) visualized a homodimeric structure, mediated by the dimerization of the UBA domain, that formed a hammer-shaped structure with IDRs wrapped around each other (Fig EV1B, Movie EV2). When each SNAP-ULK1 and SNAP-Atg1 was mixed with p62 (268-440 aa), the p62 homodimer directly bound to SNAP-ULK1 and SNAP-Atg1 via dynamic IDR-IDR and IDR-globular domain interactions (Figs 1D and E,and EV1C and D,Movies EV3 and EV4). Consistent with this, ULK1 and Atg1 directly phosphorylated recombinant p62 (268-440 aa and 320-440 aa) at Ser349 (Fig 1F). Although Ser403 was hardly phosphorylated (Fig 1F), it was also phosphorylated when mCherry-tagged full-length p62 was used (Fig 1F), indicating that the N-terminal PB1 domain of p62 is required for efficient Ser403 phosphorylation by ULK1 and Atg1. These data suggest that ULK1 directly interacts with and phosphorylates p62. A Domain structures of ULK1, Atg1, and p62. KD, kinase domain; MIT, microtubule interaction and transport domain; PB1, Phox1 and Bem1p domain; LIR, LC3interacting region; KIR, KEAP1-interacting region; UBA, ubiquitin-associated. B Three-dimensional structures of ULK1, Atg1, and p62 as predicted by Alphafold 2. C CBB staining of purified p62 (268-440 aa), p62 (320-440 aa), SNAP-Atg1, and SNAP-ULK1. D Successive HS-AFM images of p62_268-440 with SNAP-ULK1. Height scale: 0-3.5 nm; scale bar: 20 nm. E Schematics showing the molecular characteristics observed by HS-AFM. Gray spheres, globular domains consisting of N-terminal KD and C-terminal MIT domain of ULK1; pink spheres, globular domains consisting of C-terminal UBA domain of p62; blue thick solid lines, IDRs. F In vitro kinase assay. Purified recombinant p62 (268-440 aa), p62 (320-440 aa), or mCherry-p62 was incubated for 20 min at 30°C with purified SNAP-Atg1 or SNAP-ULK1 in the presence or absence of ATP. Reactions were then terminated by adding LDS sample buffer containing reducing agent, followed by immunoblot analysis with the indicated antibodies. As positive and negative controls, Huh-1 cell lysates treated with or without lambda protein phosphatase (kPP) were used. Data were obtained from three independent experiments. Asterisks show possible dimeric structures of p62 (268-440 aa), p62 (320-440 aa) and mCherry-p62. Localization of ULK1 in p62 bodies p62 undergoes LLPS upon interaction with ubiquitinated proteins in vitro, forming p62 condensates (Sun et al, 2018). We examined whether SNAP-Atg1 and SNAP-ULK1 associate with p62 condensates in vitro. Consistent with previous reports (Sun et al, 2018;Zaffagnini et al, 2018;Kageyama et al, 2021), mixing mCherry-p62 with linear octa-ubiquitin (8xUb) resulted in the formation of condensates (Fig 2A). Their size was markedly increased by phosphomimetic p62 mutations (S403E and S407E) that are known to increase the binding affinity of p62 to ubiquitin (Matsumoto et al, 2011;Pilli et al, 2012;Lim et al, 2015), compared with a slight increase in the S349E phosphomimetic mutation (Fig 2A). SNAP-Atg1 and SNAP-ULK1 were recruited to both wild-type and phosphomimetic p62 condensates when all were incubated together (Figs 2B and EV2A), but not when SNAP-tag or protein kinase A (PKA) instead of SNAP-ULK1 was used ( Fig EV2B). These results imply that both Atg1 and ULK1 specifically associate with p62 even in the droplet form. We next studied the localization of ULK1 in Huh-1 cells. Immunofluorescence analysis with an anti-ULK1 antibody showed a significant signal of ULK1 in p62 bodies, which was diminished by ULK1 depletion (Fig 2C, Appendix Fig S1). Together with FIP200, ATG13, and ATG101, ULK1 forms an initiation kinase complex for autophagosome formation (Lin & Hurley, 2016), and p62 interacts with FIP200 through the Claw domain (Turco et al, 2019), raising the possibility that the localization of ULK1 to p62 bodies is indirect and depends on the interaction of p62 with FIP200. To test this hypothesis, we developed FIP200-deficient Huh-1 cells (Appendix Fig S1). Remarkably, we observed ULK1 localization to p62 bodies even in these cells, and ULK1 signal intensity was significantly higher than in wild-type Huh-1 cells (Fig 2C), probably due to increased ULK1 protein in the FIP200-knockout cells. Exogenously expressed green fluorescent protein (GFP)-tagged ULK1 and ULK2 also localized on p62 bodies regardless of the presence of FIP200, but not in the case of GFP-LATS1, an irrelevant serine-threonineprotein kinase (Figs 2D and EV3A). An in vitro binding assay using purified p62, ULK1, and FIP200 Claw showed that ULK1 alone bound to p62 and that FIP200 did not interfere with ULK1 for p62-binding ( Fig EV3B and C). Correlative light and electron microscopy with Huh-1 cells harboring GFP-ULK1 revealed that GFP-ULK1 localizes on round structures composed of filamentous assemblies; these structures were previously identified as p62 bodies (Jakobi et al, 2020;Kageyama et al, 2021) (Fig 2E). Taken together, these data suggest that ULK1 localizes in p62 bodies through a direct ULK1-p62 interaction.

Significance of ULK1 within p62 bodies
In addition to Ser349 in KIR of p62, ULK1 phosphorylates Ser403 and Ser407 within the UBA domain of p62 (Ro et al, 2014;Lim et al, 2015). It is unclear whether the phosphorylation of the UBA domain affects Ser349 and vice versa. To address this issue, we expressed the wild-type p62, p62 S349E (S349-phosphomimetic) (Ichimura et al, 2013), p62 S349A (S349-phosphodefective) (Ichimura et al, 2013), p62 S403E S407E (S403E and S407E-phosphomimetic), or p62 S403A S407A (S403A and S407A-phosphodefective) mutant in p62deficient Huh-1 cells and investigated the phosphorylation states of Ser349, Ser403, and Ser407. As shown in Appendix Fig S2, while the p62 S349E mutant had no effect on the phosphorylation of the UBA domain, the p62 S403E S407E mutant enhanced the phosphorylation of Ser349. Considering that the phosphorylation of S403 and S407 is thought to promote the LLPS of p62, it is plausible that Ser349 phosphorylation of p62 occurs within p62 bodies.
Next, to investigate the significance of ULK1 and ULK2 within p62 bodies, we utilized MRT68921, which is the most potent inhibitor of ULK1 and ULK2, with IC 50 values of 2.9 and 1.1 nM, respectively (Petherick et al, 2015). As predicted, the treatment of Huh-1 cells with 2.5 lM MRT68921 decreased not only the level of phosphorylated ATG13, but also those of the Ser349-and Ser403phosphorylated p62 forms ( Fig 3A). Even when we used a lower concentration of MRT68291 (1 lM), the levels of the Ser349-and Ser403-phosphorylated p62 forms decreased to the same degree ( Fig EV4A). Similar results were obtained with ULK-101, another inhibitor of ULK1 and ULK2 (Martin et al, 2018) (Fig EV4A).
We observed extensive colocalization of the Ser403phosphorylated form in p62 bodies ( Fig 3B). The signal intensity of Ser403-phosphorylated p62 in p62 bodies became weaker when ▸ Figure 2. Localization of ULK1 on p62 bodies.
Source data are available online for this figure.
4 of 20 Huh-1 cells were treated with MRT68921 ( Fig 3B). MRT68921 treatment slightly but significantly decreased both the size and number of p62 bodies ( Fig 3B). These results suggest that while ULK1 and ULK2 contribute to the LLPS of p62 through the phosphorylation of Ser403 of p62, the dephosphorylation of Ser403 within p62 bodies hardly has an effect on already formed p62 bodies. Next, we tested whether inhibition of ULK1 and ULK2 affected KEAP1-localization within p62 bodies. Huh-1 cells were cultured in the presence or absence of MRT68921 and immunostained with anti-p62 and anti-Ser349-phosphorylated p62-specific antibodies. The p62 bodies in Huh-1 cells contained the Ser349-phosphorylated form ( Fig 3C). Upon exposure to MRT68921, the signal intensities of phosphorylated p62 on p62 bodies markedly decreased ( Fig 3C). Double immunofluorescence analysis with anti-p62 and anti-KEAP1 antibodies showed extensive localization of KEAP1 in the p62 bodies ( Fig 3C). The signal intensity of KEAP1 in the bodies was significantly attenuated by treatment with MRT68921 ( Fig 3C), suggesting release of KEAP1 from the bodies to the cytoplasm and subsequent NRF2 inactivation. Indeed, the gene expression of NRF2 targets such as glutamate-cysteine ligase catalytic subunit (GCLC), NAD(P)H quinone dehydrogenase 1 (NQO1), UDP-glucose 6-dehydrogenase (UGDH), superoxide dismutase 1 (SOD1), and p62 itself was decreased by MRT68921 or ULK-101 treatment (Figs 3D and EV4B).
These data suggest that ULK1 and ULK2 in p62 bodies contribute to the activation of NRF2 by phosphorylating p62 at Ser349 and promoting sequestration of KEAP1 within p62 bodies.

Dynamics of KEAP1 in S349-phosphorylated p62 bodies
In the next series of experiments, we sought to determine whether Ser349 phosphorylation of p62 affects KEAP1 dynamics in p62 bodies. To do this, we generated p62 KEAP1 double-knockout Huh-1 cells (Appendix Fig S1) and expressed GFP-tagged wild-type p62, phosphomimetic p62 S349E (Ichimura et al, 2013), phosphodefective p62 S349A (Ichimura et al, 2013), or KEAP1 interaction-defective p62 T350A (Komatsu et al, 2010) together with mCherry or mCherrytagged KEAP1. The fluorescence analysis revealed that in the absence of mCherry-KEAP1, wild-type GFP-p62 and all GFP-p62 mutants formed round, liquid droplet-like structures ( Fig 4A). When we coexpressed wild-type GFP-p62 or the mutants with mCherry-KEAP1 in the double knockout Huh-1 cells, mCherry-KEAP1 colocalized well with GFP-p62-positive structures except those composed of GFP-p62 T350A (Fig 4A). We measured the circularity of each GFP-p62-positive structure composed of wild-type p62 or the mutants in the presence or absence of mCherry-KEAP1. Circularity values close to 1 were associated with liquid droplets, while lower values ◀ Figure 3. Significance of p62 phosphorylation by ULK1 and ULK2.
A Immunoblot analysis. Huh-1 cells were treated with or without 2.5 lM MRT68921 for 6 h, and the cell lysates were subjected to immunoblot analysis with indicated antibodies. The asterisk indicates non-specific bands. Data shown are representative of three separate experiments. Bar graphs show the results of quantitative densitometric analysis of Ser349-or Ser403-phosphorylated p62 forms relative to total p62 (n = 3), and of Ser318-phosphorylated ATG13 relative to total ATG13 (n = 3). Data are means AE s.e. Statistical analysis was performed by Welch's t-test. B Immunofluorescence microscopy. Huh-1 cells were treated with or without 2.5 lM MRT68921 for 6 h and immunostained with the indicated antibodies. The ratio of p62 (p-S403) to p62 on p62 bodies and the size and number of p62 bodies in each cell were quantified (n = 500 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25 th -75 th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 lm (main panels), 1 lm (inset panels). C Immunofluorescence microscopy. Huh-1 cells were treated with or without 2.5 lM MRT68921 for 6 h and immunostained with the indicated antibodies. The ratio of p62 (p-S349) to p62 and the signal intensity of KEAP1 on p62 bodies in each cell were quantified (n = 500 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25 th -75 th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 lm (main panels), 1 lm (inset panels). D Gene expression of NRF2 targets. Total RNAs were prepared from Huh-1 cells treated with or without 2.5 lM MRT68921 for 6 h. Values were normalized against the amount of mRNA in non-treated Huh-1 cells. qRT-PCR analyses were performed as technical replicates on each biological sample. Data are means AE s.e. Statistical analysis was performed by two-sided Welch's t-test.
Source data are available online for this figure.
A Fluorescence microscopy. GFP-p62, GFP-p62 S349E , GFP-p62 S349A , or GFP-p62 T350A were co-transfected with mCherry or mCherry-KEAP1 into Huh1 p62 KEAP1 doubleknockout cells. Twenty-four hours after transfection, the fluorescence images were observed. B Circularity of p62 bodies. The circularity of p62 bodies in each cell was quantified (n = 150 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25 th -75 th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by two-sided Welch's t-test. Scale bar: 2 lm. C The ratio of the mean signal intensity of mCherry-KEAP1 on p62 bodies to that of the cellular area in each type of Huh-1 p62 KEAP1 double-knockout cell expressing mCherry-KEAP1 (n = 150 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25 th -75 th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Sid ak's test after one-way ANOVA. D In vitro formation of p62-KEAP1-8xUb condensates. 5 lM SNAP-KEAP1 labeled with SNAP-Surface Alexa Fluor 488 was premixed with 10 lM mCherry-p62 wild-type, or mCherry-p62 mutants before mixing with 10 lM SNAP(649)-8xUb. Scale bars: 20 lm. E FRAP analyses of mCherry-KEAP1 localized in p62 bodies comprised of GFP-p62 S349E or GFP-p62 S349A . The half-time of recovery (t50) and mobile fraction (MF) of mCherry-KEAP1 was measured by FRAP of whole p62 bodies (n = 7). Data are means AE s.d. Statistical analysis was performed by two-sided Welch's t-test. F FLIP analyses of mCherry-KEAP1 localized in p62 bodies comprised of GFP-p62 S349E or GFP-p62 S349A . The fluorescence loss of mCherry-KEAP1 in GFP-p62 bodies (n = 14) was measured 30 min after photobleaching over a large area of cells. G FRAP analyses of mCherry-KEAP1 localized in p62 bodies comprised of GFP-p62 S349E or GFP-p62 S349A . t50 and MF of mCherry-KEAP1 were measured by FRAP of the central portions of p62 bodies (n = 10). Data are means AE s.d. Statistical analysis was performed by two-sided Welch's t-test.
Source data are available online for this figure. correlated with gels or aggregates (Strom et al, 2017;Faruk et al, 2021a). In the absence of mCherry-KEAP1, all GFP-p62positive structure values were close to 1 (Fig 4B), suggesting that they were liquid droplets. Remarkably, the circularity of GFP-p62 S349E bodies but not others was significantly decreased when these bodies colocalized with mCherry-KEAP1 (Fig 4B). We also measured the signal intensity of mCherry-KEAP1 on p62 bodies consisting of wild-type p62 or a series of different mutants and found that the intensity of mCherry-KEAP1 on p62 S349E bodies was higher than that on wild-type p62 and p62 S349A bodies ( Fig 4C).
Meanwhile, there was no significance difference in mCherry-KEAP1 intensity between wild-type p62 and p62 S349A bodies (Fig 4C). Furthermore, an in vitro LLPS assay revealed that introduction of the S349E mutation in p62 wild-type or p62 S403E S407E resulted in the formation of amorphous aggregates rather than liquid droplets, but only in the presence of KEAP1 (Fig 4D, Appendix Fig S3). These data suggest that strong binding of KEAP1 to p62 as a result of the S349E mutation changes the biophysical properties of p62 bodies. We hypothesized that once mCherry-KEAP1 was incorporated into S349-phosphorylated p62 bodies, KEAP1 efflux from the bodies would be significantly reduced due to the close interaction between the phosphorylated p62 and KEAP1. To prove this, we used fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) (Ishikawa-Ankerhold et al, 2012) to evaluate KEAP1 influx and efflux into p62 bodies consisting of GFP-p62 S349E or p62 S349A . To measure the influx of mCherry-KEAP1 into GFP-p62 bodies, the whole fluorescence of mCherry-KEAP1 in GFP-p62 S349E or p62 S349A bodies was photobleached, and the fluorescence recovery was measured. It is common to characterize molecular dynamics in FRAP experiments by the half-time of recovery (t50) and the mobile fraction (Reits & Neefjes, 2001). Based on these parameters, the influx of mCherry-KEAP1 from the surrounding environment was comparable between p62 S349E and p62 S349A bodies (Fig 4E, Movies EV5 and EV6). Next, to examine the efflux of mCherry-KEAP1 from GFP-p62 bodies, we carried out FLIP analysis. When approximately 80% of the cellular region is photobleached, the fluorescent signal in the cytoplasm is transiently reduced, followed by a gradual recovery due to influx from the nonbleached region. If there is an outflow of mCherry-KEAP1 from p62 bodies, the fluorescence intensity of mCherry-KEAP1 within p62 bodies in the non-bleached area should decrease after photobleaching. While the signal intensity of mCherry-KEAP1 in p62 S349A bodies decreased to about 58.2 AE 0.05% of the baseline value at 30 min after photobleaching, it remained higher (72.0 AE 0.07%) in the case of p62 S349E bodies (Fig 4F, Movies EV7 and EV8). Finally, to investigate the inner fluidity of mCherry-KEAP1 in GFP-p62 bodies, we measured the fluorescence recovery of mCherry-KEAP1 after photobleaching of the central portion of GFP-p62 bodies. The t50 of mCherry-KEAP1 in p62 S349E bodies was 8.51 AE 4.83 min, which was much slower than that seen with p62 S349A bodies (3.32 AE 1.23 min) (Fig 4G, Movies EV9 and EV10). Taken together, these results suggest that Ser349 phosphorylation of p62 results in the retention of KEAP1 in p62 bodies, and when this sequestration is prolonged, the inner fluidity of these bodies is decreased.

Physiological significance of Ser349 phosphorylation of p62 in mice
To clarify the physiological role of Ser349 phosphorylation of p62 in vivo, we generated knock-in mice that expressed p62 in which Ser351 (corresponding to human Ser349) was replaced by Glu (p62 S351E/+ mice) or Ala (p62 S351A/+ mice). Initially, we tried to use the CRISPR/Cas9 system to generate both knock-in mice, but could obtain only p62 S351A/+ mice. We therefore attempted to use prime editing, a recently developed system, to generate p62 S351E/+ mice using mouse embryonic stem cells (mES cells) (Anzalone et al, 2019). Even with this method, however, we were unable to obtain chimeric mice with high chimerism. We did succeed in generating a male chimeric mouse with low chimerism and germline transmission. In vitro fertilization using sperm from the chimeric mouse was performed to obtain a sufficient number of heterozygotes for the experiments. The resulting p62 S351E/+ mice showed severe growth retardation and mild hepatomegaly at P12 and P15 (Fig 5A-C), which is probably why knock-in mice could not be obtained by the above method. RNAseq analysis of wild-type and p62 S351E/+ mouse livers demonstrated increased gene expression of NRF2 targets (Fig 5D). Consistent with these results, real-time PCR analysis showed that the gene expression of NRF2 targets such as glutathione S-transferase Mu 1 (Gstm1), Nqo1, Ugdh, and p62 was much higher in the liver of p62 S351E/+ mice than in wild-type mice (Fig 5E). We also found that the levels of GSTM1, NQO1, and UGDH, and the nuclear level of NRF2, were markedly higher in ▸ Figure 5. Physiological significance of p62 phosphorylation at S351 in mice.
Source data are available online for this figure. 10 of 20 The EMBO Journal 42: e113349 | 2023 Ó 2023 The Authors p62 S351E/+ mice than in wild-type mice (Fig 5F). KEAP1 is incorporated into p62 bodies (Kageyama et al, 2021), and the complex of KEAP1 and p62 is degraded by autophagy (Taguchi et al, 2012). Therefore, the phosphorylation of p62 at Ser349, which increases the binding affinity to KEAP1, should promote the degradation of KEAP1. In fact, the level of KEAP1 was lower in p62 S351E/+ mice compared with that in wild-type mice (Fig 5F). These results indicate that the expression of p62 S351E at half the level of endogenous p62 is sufficient for KEAP1 inactivation and subsequent NRF2 activation in vivo. Anatomical analysis revealed that the forestomach wall of p62 S351E/+ mice was obviously thickened compared with that of wild-type mice (Appendix Fig S4). Hematoxylin and eosin (HE) staining indicated that the esophagus and forestomach of p62 S351E/+ mice had a remarkably thicker stratum corneum than those of wildtype mice, but such differences were not observed in the skin (Fig 5G). In the forestomach, the epithelial layers below the stratum corneum were slightly thicker in p62 S351E/+ mice than in wild-type mice, but this was not evident in the esophagus. Immunohistochemistry (IHC) revealed that in the epithelia of the esophagus and forestomach, the intensity of NQO1 was higher in p62 S351E/+ mice than in wild-type mice (Fig 5H). The staining of the cell proliferation marker Ki-67 showed no significant difference between mutant and wildtype organs (data not shown). Serum data from p62 S351E/+ mice indicated a slightly but significantly elevated aspartate aminotransferase level, signs of malnutrition (low blood glucose and high cholesterol), and signs of dehydration (increased blood urea nitrogen and creatinine) (Fig 5I). These results strongly suggested that p62 S351E/+ mice are a phenocopy of Keap1-deficient mice (Wakabayashi et al, 2003) and that impaired nutritional intake due to hyperkeratosis in the esophagus and forestomach could be the primary cause of the severe phenotype of p62 S351E/+ mice. In striking contrast to these mice, p62 S351A/+ and p62 S351A/S351A mice were fertile and showed no obvious growth retardation (Fig EV5A-C). Morphological and biochemical analyses indicated no differences in phenotypes (including NRF2 activation) between wild-type and p62 S351A/S351A mice (Fig EV5D-G), implying that Ser351 phosphorylation of p62 is unnecessary for mouse development and survival. Taken together, these results indicate that Ser351 phosphorylation of p62 is physiologically important for the control of NRF2 activity in vivo, most likely by regulating the redox-independent KEAP1-NRF2 pathway.

Discussion
The ULK1 kinase complex functions as the most upstream factor in autophagosome formation (Noda & Fujioka, 2015;Lin & Hurley, 2016). In Saccharomyces cerevisiae, nutrient deprivation causes phosphatase-induced dephosphorylation of Atg13, which results in the formation of higher order structures of the Atg1 kinase complex (the ULK1 kinase complex in mammals) and subsequently the formation of a liquid-droplet, pre-autophagosomal structure (Yamamoto et al, 2016;Fujioka et al, 2020). Nutrient starvation activates the ULK1 kinase complex, which phosphorylates ATG proteins, including Beclin 1 (Russell et al, 2013), ATG14 (Park et al, 2016;Wold et al, 2016), and ATG9 (Papinski et al, 2014), and contributes to the initiation of autophagosome formation (Noda & Fujioka, 2015;Lin & Hurley, 2016). On the contrary, ULK1 phosphorylates substrates that are not directly involved in autophagosome formation, such as SEC16A (Joo et al, 2016), glycolytic enzymes , and STING (Konno et al, 2013). p62 is also a member of this category and is closely involved in LLPS, and the p62 body formation (Sanchez-Martin et al, 2019). In this study, we showed that ULK1 localized to p62 bodies (Fig 2) and phosphorylated Ser349 of p62 (Fig 1), the latter of which is required for KEAP1 localization and retention to p62 bodies and subsequent NRF2 activation (Figs 3 and  4). Thus, ULK1 modulates the formation and degradation of p62 bodies and also plays a role in the antioxidative-stress response. When does ULK1 phosphorylate p62? Since ULK1 was localized in p62 bodies both in vitro and in vivo (Fig 2), it is plausible that ULK1 phosphorylates Ser349 of p62 located in p62 bodies. Indeed, we observed increased S349-phosphorylation in the p62 S403E S407E mutant, which promotes the LLPS of p62 (Appendix Fig S2). How does ULK1 recognize p62 bodies? Our HS-AFM experiments showed that the p62 homodimer directly bound to ULK1 via dynamic IDR-IDR and IDR-globular domain interactions (Fig 1D). It is known that IDRs specifically interact with multiple target molecules through a binding mode called "coupled folding and binding" (Sugase et al, 2007). This binding mode may facilitate conformational changes and phosphorylation of large numbers of p62 molecules in p62 bodies, because substitutions between IDRs that bind to target molecules occur very rapidly (Wright & Dyson, 2015). There is no highly conserved amino acid sequence region among the IDRs of ULK1, ULK2, and Atg1 (data not shown). As shown by HS-AFM analysis (Fig 1D), IDR-mediated interaction between ULK1 and p62 is transient and rather weak. In such cases, it is generally difficult to find conserved binding sequences and motifs due to the extremely low sequence identity. With the phosphomimetic p62 mutant p62 S349E , influx of KEAP1 into p62 bodies predominated over efflux (Fig 4E and F). The coexpression of p62 S349E and KEAP1 in p62 KEAP1 double-knockout Huh-1 cells reduced the circularity of p62 bodies compared with the expression of p62 S349E alone (Fig 4A and  B), and also decreased the fluidity of KEAP1 molecules within p62 bodies (Fig 4G). When the sequestration of KEAP1 within p62 bodies surpasses a certain threshold level, these bodies convert from liquid-like to gel-like droplets. What does this mean? Since autophagy is known to target gel-like rather than liquid-like droplets (Zhang et al, 2018;Yamasaki et al, 2020;Kageyama et al, 2021), it is possible that incorporation of a certain number of KEAP1 molecules into p62 bodies changes them into gel-like aggregates and enhances autophagic degradation. This is consistent with two findings: adding KEAP1 to p62 condensates consisting of p62 S349E resulted in amorphous aggregates in vitro (Fig 4D), and p62 S351E/+ mice had reduced levels of not only Ser403-phosphorylated p62 representing p62 bodies but also KEAP1 (Fig 5F). Once p62 bodies are degraded by autophagy, the interaction between Ser349phosphorylated p62 and KEAP1 should be suppressed because Ser349 phosphorylation of p62 occurs mainly in p62 bodies. As a result, KEAP1 remains in the cytoplasm, and NRF2 is degraded. In other words, the retention of KEAP1 in p62 bodies above a threshold level is thought to suppress NRF2 activation in a feedback regulation process.
Nrf2 À/À mice grow normally and are fertile (Itoh et al, 1997), though they are susceptible to oxidative stress and reactive electrophiles (Yamamoto et al, 2018). In addition, they show tooth decolorization due to defective iron transport in the enamel (Yanagawa et al, 2004), which makes them easily distinguishable from wild- The EMBO Journal 42: e113349 | 2023 type and heterozygous mice. p62 S351A/S351A , the phosphodefective p62 knock-in mice in which p62-mediated NRF2 activation should be impaired, were also fertile and did not differ from wild-type mice for 1 year, at least under specific pathogen-free conditions ( Fig EV5). However, their incisors were brownish-yellow, in contrast to the grayish white incisors in Nrf2-deficient mice (data not shown), indicating that p62 S351A/S351A mice retain NRF2 activity. p62-mediated NRF2 activation was not required for development or survival, at least under steady-state conditions. The redoxindependent antioxidative stress response might have an anti-aging effect since p62 bodies are thought to increase when the activities of both autophagy-lysosomes and the ubiquitin-proteasome system decrease with aging. Further research is needed to determine whether the redox-independent stress response is activated with aging, and if so, which tissues are affected.
In sharp contrast to p62 S351A/S351A mice, the phosphomimetic p62 knock-in mice p62 S351E/+ , in which p62-mediated NRF2 activation is persistently activated, showed severe phenotypes. The mice had impaired nutritional intake due to hyperkeratosis in the esophagus and forestomach, which led to malnutrition and dehydration (Fig 5G-I). This represented almost a phenocopy of Keap1-deficient mice, which exhibit hyperactivation of NRF2 and show hyperkeratosis in the esophagus and forestomach (Wakabayashi et al, 2003). One difference is that p62 S351E/+ mice do not develop skin hyperkeratosis, which is observed in Keap1-knockout mice (Wakabayashi et al, 2003). Why does constant activation of NRF2, which has an inherently cytoprotective role, cause a severe phenotype? The esophagus and stomach are exposed to a variety of toxic foods and drinks. This may cause wounds that require NRF2 for healing, and a redox-independent p62-mediated pathway may be at work. Meanwhile, the healing of skin wounds may be mediated by redoxdependent NRF2 activation (Braun et al, 2002). It is plausible that transient activation of NRF2 in response to toxicity, whether redox dependent or independent, is important for biological defense, and that persistent activation leads to inordinate defense responses (e.g., excessive keratinization). In the case of redox-independent stress responses, transient activation of NRF2 is presumably regulated by phosphorylation, dephosphorylation, and autophagic degradation of p62 body.
In conclusion, we showed for first time that the redoxindependent NRF2 activation pathway, which is mediated by p62 bodies and their phosphorylation, is physiologically important.

Sample preparation for HS-AFM imaging
For imaging of SNAP-ULK1 and p62_268-440, SNAP-ULK1 (50 nM) or p62_268-440 (10 nM) was deposited onto freshly cleaved mica glued to the top of a glass stage (diameter, 1.5 mm; height, 2 mm). After incubation for 3-5 min, the mica was rinsed and immersed in the liquid cell containing~90 ll of imaging buffer A (20 mM NaCl, 20 mM HEPES-NaOH [pH 7.5], 1 mM MgCl 2 , 0.1 mM ATP) or imaging buffer B (20 mM NaCl, 20 mM HEPES-NaOH [pH 7.5], 1 mM MgCl 2 ), respectively. For imaging of p62_268-440 with SNAP-ULK1 or SNAP-Atg1, 25 nM p62_268-440 and 50 nM SNAP-ULK1 or 10 nM p62_268-440 and 5 nM SNAP-Atg1 in imaging buffer A were mixed in a 0.5-ml tube. The mixed protein solution was deposited onto freshly cleaved mica and incubated for 3-5 min. After rinsing with imaging buffer A, the mica was immersed in~90 ll of imaging buffer A.

In vitro LLPS assay
Fluorescence microscopy-based competitive binding assay GST-accept beads (particle size 50-150 lm) was selected as the beads to be coated by GST-tagged mCherry-p62. 2 ll of 50% suspension of GST-accept beads in a PCR tube was washed with 20 ll of Buffer F (150 mM NaCl, 20 mM HEPES-NaOH pH 7.4) three times. Then, 5 lM GST-mCherry-p62 was incubated with the beads in 5 ll of Buffer F for 30 min. After five times washing with 20 ll of Buffer F, 0.4 lM SNAP-ULK1 labeled with Alexa Fluor 488 and 0-40 lM MBP-FIP200 Claw labeled with Alexa Fluor 647 C2 maleimide were incubated with the beads in 5 ll of Buffer F containing 1 mM DTT for 1 h. After five times washing with 20 ll of Buffer F containing 1 mM DTT, the beads were resuspended with 20 ll of Buffer F containing 1 mM DTT. 6 ll of the beads suspension on glass-bottom dish (MatTek) was then imaged using an FV3000RS confocal laser-scanning microscope (Olympus) at~23°C. Fluorescence intensities of ULK1 and p62 on the beads were measured and analyzed by Fiji (ImageJ).

Correlative light and electron microscopic analysis
Huh-1 cells on coverslips etched with 150-lm grids (CS01885, Matsunami Glass Ind. Osaka, Japan) were transfected with pMRX-IP-GFP-ULK1 or pMRX-IP-GFP-ULK2. After 24 h, cells were fixed with 2% PFA-0.1% glutaraldehyde (GA) in 0.1 M PB (pH 7.4). Then, phase-contrast and fluorescence images were obtained using a confocal microscope (FV1000). After image acquisition, the cells were fixed again with 2% PFA and 2% GA in 0.1 M PB (pH 7.4), processed according to the reduced-osmium method (Arai & Waguri, 2019), and embedded in Epon812. Areas containing cells of interest were trimmed, cut as serial 80-nm sections, and observed using an electron microscope (EM; JEM1400; JEOL, Tokyo, Japan). Light microscopy and EM images were aligned according to three p62 bodies using Photoshop CS6 (Adobe).

Histological analyses
Mouse livers were excised, cut into small pieces, and fixed by immersion in 4% PFA-4% sucrose in 0.1 M PB, pH 7.4. After rinsing, they were embedded in paraffin for immunostaining. Paraffin sections of 3-lm thickness were prepared and processed for HE staining or IHC. For IHC, antigen retrieval was performed for 20 min at 98°C using a microwave processor (MI-77, AZUMAYA, Tokyo, Japan) in 1% immunosaver (Nissin EM, Tokyo, Japan). Sections were blocked and incubated for 2 days at 4°C with the following primary antibodies: rabbit polyclonal antibody against NQO1 (Abcam), followed by N-Histofine simple stain mouse MAX PO kit (NICHIREI BIOSCIENCES, Tokyo, Japan) using 3,39diaminobenzidine. Images of the stained specimens were acquired with a microscope (BX51, Olympus) equipped with a cooled CCD camera system (DP-71, Olympus).
FLAP and FLIP assays p62/KEAP1 double-knockout Huh-1 cells expressing GFP-p62 S349E or GFP-p62 S349A in a doxycycline treatment-dependent manner were generated using a reverse tet-regulated retroviral vector, as previously reported. To induce the expression of GFP-p62 S349E or GFP-p62 S349A , the cells were treated with 50 ng/ml of doxycycline (Dox, Sigma-Aldrich) for 24 h. Thereafter, m Cherry-KEAP1 was transfected with Lipofectamine 3000 (Thermo Fisher Scientific) and cultured for 24 h. In FRAP assays, GFP-p62 bodies that were positive for mCherry-KEAP1 were bleached using a laser intensity of 10% at 488 nm, and then the fluorescence recovery of mCherry was recorded. In FLIP assays, around 80% of the total cell area was set as the region of interest for photobleaching (excitation output level: 10% at 561 nm; iterations: 3-5) using FV31S-SW software (Olympus), and then the fluorescence loss of mCherry-KEAP1 in GFP-p62 S349E or GFP-p62 S349A bodies was recorded. The fluorescence intensity of mCherry-KEAP1 within the p62 bodies in the photobleaching area recovered to 32% at 15 min after photobleaching and reached equilibrium. Therefore, the fluorescence intensity of mCherry-KEAP1 within the p62 bodies in the nonphotobleaching area was measured at 30 min after fluorescence loss. Olympus FV31S-SW software (version: 2.4.1.198) and cellSens Dimension Desktop 3.2 (Build 23706) was used for image collection and analysis. The mobile fraction was calculated from 10 measurements by the following equation: Mf = (F∞ À F0)/(Fi À F0), where Mf is the mobile fraction, F∞ is the fluorescence intensity after full recovery (plateau), Fi is the initial fluorescence intensity prior to bleaching, and F0 is the fluorescence intensity immediately after bleaching. The half-time (t50) of fluorescence recovery was calculated from 10 measurements by curve fitting using the one-phase decay model of GraphPad PRISM 9 (GraphPad Software, San Diego, CA, USA).

RNA sequencing (RNA-seq)
Total RNA from livers of p62 +/+ and p62 S351E/+ mice at P19 was extracted using the RNeasy Mini Kit (Qiagen, Hulsterweg, Netherlands). Ribosomal RNA was depleted using a NEBNext rRNA Depletion Kit (NEB). For sequencing, a cDNA library was synthesized using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB). Sequencing was performed on a NextSeq 500 sequencer (Illumina, San Diego, CA, USA) with 75-bp single-end reads. Resulting reads were mapped to the UCSC (University of California, Santa Cruz, CA, USA) mm10 reference genome using the Spliced Transcripts